Zn is an essential nutrient but can be toxic if overaccumulated. Therefore, organisms have homeostatic mechanisms to maintain adequate intracellular supplies of Zn and prevent overaccumulation as extracellular levels change. The long-range goal of this research is to understand the molecular mechanisms of Zn homeostasis in eukaryotic cells. This process involves Zn uptake and efflux transporters in the plasma membrane, organellar transporters that move Zn into and out of intracellular compartments, intracellular Zn binding proteins and chelators, and the regulatory systems that control their activities. Despite the nutritional importance of Zn, we still know little about Zn homeostasis. This proposal describes a combined genetic, molecular, biochemical, and biophysical approach to the study of Zn homeostasis in the yeast Saccharomyces cerevisiae. This organism has been a useful model for understanding many biological processes and is proving to be equally invaluable for the study of Zn. The expression of >40 genes in the yeast genome is induced in Zn-deficient cells by Zap1, a Zn-responsive transcriptional activator. Zap1 is the intracellular Zn sensor responsible for this regulation and its activity is modulated by Zn binding directly to the protein. Zap1 has several identified functional domains. At its C-terminus is a DNA binding domain (DBD). This domain binds specifically to sites found in the promoters of Zapl's target genes. Zapl also contains two activation domains, AD1 and AD2, both of which activate transcription. Our results indicate that Zap1 is regulated by Zn via three, possibly even four, different mechanisms. First, Zap1 controls its own expression through positive transcriptional autoregulation. This autoregulation controls the level of Zapl protein in the cell and ZRE binding. In addition, Zn independently controls the activities of AD1 and AD2. Thus, regulation of Zap1 activity by Zn requires two independent Zn sensors to control these activities. Our preliminary studies have led us to hypotheses regarding these sensors and the molecular mechanisms underlying these regulatory events. These hypotheses will be tested in this proposed research. The fourth mechanism we will assess is if DBD function per se is Zn regulated. These studies will lead to fundamental insights into metallosensing and Zn homeostasis.